Project Summary Tumor cells have increased rates of proliferation that demand enhanced energy and biomass requirements. Proliferating tumor cells commonly support macromolecule duplication by shifting bioenergetics from oxidative phosphorylation, as observed in non-proliferating cells, to aerobic glycolysis as this form of metabolism produces critical building blocks and energy. Tumor cell lipid metabolism is intimately linked to aerobic glycolysis and recent findings suggest a critical role for lipid metabolism in tumorigenesis and malignant progression. Fatty acid synthase (FASN), the key enzyme in endogenous lipid synthesis, is overexpressed in breast cancer and is linked to poor prognosis. Yet, despite the importance of lipid metabolism in breast cancer, it is an understudied area. One way that lipid metabolism is regulated is by receptor tyrosine kinase (RTK) signaling. Dysregulation of RTK signaling can promote aberrant metabolism that contributes to cellular transformation. Therefore, it is necessary to elucidate the mechanisms that promote malignancy and metabolic reprogramming. We recently found that ephrin-A1, a prototypic ligand of EphA2 RTK, suppresses mammary gland hyperplasia. We next performed two separate experiments to evaluate whether this growth phenotype was due to the intrinsic function of ephrin-A1 within the epithelial cells or the surrounding stroma. First, we isolated mammary epithelial cells from both WT and ephrn-A1 knockout mice and performed 3-dimensional organoid cultures. Next, we performed mammary gland transplant procedures where we transplanted mammary glands into the cleared fat pads of mice of opposite genotype. Both of these experiments indicated that it is the function of ephrin-A1 within epithelial cells that plays the greatest role in this growth phenotype. Since lipid metabolism can influence growth, we tested whether ephrin-A1 regulated levels of cellular lipid deposits. In human cancer cell lines, we applied shRNA techniques to knockdown ephrin-A1 (EFNA1) and observed that ephrin-A1 knockdown cells not only had elevated levels of lipid deposits but also acquired invasive properties and enhanced tissue spheroid formation. Since FASN contributes to growth, is linked to breast cancer progression and is the key enzyme in endogenous lipid synthesis, we investigated FASN protein expression in hyperplastic glands/tumors from a small number of WT and ephrin-A1 knockout mice. We were able to demonstrate that ephrin-A1 knockout mice have elevated levels of FASN protein compared to WT tissues. These preliminary data suggest that ephrin-A1 may regulate breast tumor malignancy by regulating lipid metabolism. To further investigate this in the context of human disease, invasive ductal carcinoma and lymph node metastases tissue microarray datasets were analyzed for ephrin- A1 and FASN protein expression. We observed that low ephrin-A1 expression correlated with elevated FASN protein expression. Based on our preliminary data, we hypothesize that ephrin-A1 suppresses mammary epithelial cell lipid metabolism to inhibit breast cancer development and progression. To elucidate the mechanistic connection between ephrin-A1, lipid metabolism and breast cancer growth we have proposed experiments in 2 Specific Aims. In Aim 1, we will determine the mechanism for ephrin-A1 regulation of lipid metabolism in tumor cells and will determine whether it is through EphA2/HER2 clustering or the Akt-mTOR-SREBP signaling. Based on previous studies, we postulate that ephrin-A1 binding to EphA2 RTK may prevent EPHA2/HER clustering or may promote its degradation thus inhibiting lipid biosynthesis. Another possibility is the ephrin-A1 suppresses SREBP activation, since we have shown that ephrin-A1 inhibits Akt activity in breast epithelial cells. In Aim 2 we will systematically analyze ephrin-A1 function in mammary tumor development and progression in MMTV-Neu mice. From these animals we will evaluate protein expression within tumors and will be able to further evaluate targets identified in aims 1. Together these aims will identify the role of ephrin-A1 in breast cancer development, progression and metabolism. Furthermore, these studies will have broader implications since it may reveal a novel mechanism of metabolic regulation, potentially linking ephrin-A1 to other cancers and human diseases with aberrant lipid metabolism.